Bottom Line:
Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome.Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process.Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers.

ABSTRACTThe adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.

f4: Dimeric Grb2 inhibits while monomeric Grb2 promotes MAP kinase activity.(a) Stable HEK293T cells overexpressing FGFR2–GFP (as control, C) and strep-tagged wild-type Grb2 or the dimerization-defective Y160E mutant were stimulated with 50 ng/ml FGF2 or EGF for 15 and 5 min, respectively. Cell-lysates were analysed for phospho-ERK (pERK), total ERK and Grb2 expression levels using specific antibody and Odyssey infra-red imaging. (b) HEK293T cells overexpressing FGFR2–GFP with indicated strep-tagged Grb2 in serum were lysed and subjected to strep-tactin affinity purification. The resulting co-precipitated complexes along with input cell-lysates were analysed for SOS and FRS2 binding using respective antibody. The immunoblot was also re-probed for Grb2 as a loading control. The data presented are representative of three independent experiments.

Mentions:
We have shown two independent mechanisms of Grb2 dissociation, both dependent on tyrosine kinase activity. Both these mechanisms could play a fundamental role in Grb2-mediated intracellular signalling. Since Grb2 is known to bind to its downstream effector SOS1, we postulate that only mGrb2 elicits downstream effector function and hence the dimeric Grb2 (dGrb2) is incapable of signal transduction. We tested this hypothesis by investigating the downstream signalling effects of mGrb2 versus dGrb2 in mammalian cells. In cells overexpressing high concentrations the population of Grb2 is expected to be predominantly dGrb2, whilst the dimerization-defective Grb2 mutants will be monomeric regardless of their intracellular concentrations. Cells were transfected with WTGrb2 and Y160EGrb2, and downstream MAPK signalling was compared. The results show that overexpression of WTGrb2 leads to a significant decrease in FGF2- and EGF-induced extracellular signal-regulated kinases (ERK) phosphorylation. An equivalent level of dimerization-defective mutant overexpression on the other hand retains ligand-induced ERK activity (Fig. 4a and Supplementary Fig. 5a). We also confirmed Grb2 concentration-dependent MAPK inhibition by serially increasing in the intracellular Grb2 protein concentration in cells. A direct correlation between increased Grb2 concentration and decreased MAPK was observed (Supplementary Fig. 5c–e). Since the formation of the Grb2–SOS complex is pivotal for RTK-stimulated Ras/MAP kinase activation, we investigated whether the differential complex formation by dGrb2 versus mGrb2 with SOS1 could explain the observed suppression of ERK phosphorylation. Grb2 was affinity purified from cells and the resulting co-precipitating complex was analysed for SOS1 (Fig. 4b and Supplementary Fig. 5b). The results show that the monomeric Y160EGrb2 and N188/214DGrb2 form stable complexes with SOS1 whereas the dimeric WTGrb2 does not. As a control the immunoblot was re-probed with anti-FRS2 antibody, which showed that both dGrb2 and mGrb2 were equally proficient in precipitating the upstream adaptor protein required for FGF receptor signalling.

f4: Dimeric Grb2 inhibits while monomeric Grb2 promotes MAP kinase activity.(a) Stable HEK293T cells overexpressing FGFR2–GFP (as control, C) and strep-tagged wild-type Grb2 or the dimerization-defective Y160E mutant were stimulated with 50 ng/ml FGF2 or EGF for 15 and 5 min, respectively. Cell-lysates were analysed for phospho-ERK (pERK), total ERK and Grb2 expression levels using specific antibody and Odyssey infra-red imaging. (b) HEK293T cells overexpressing FGFR2–GFP with indicated strep-tagged Grb2 in serum were lysed and subjected to strep-tactin affinity purification. The resulting co-precipitated complexes along with input cell-lysates were analysed for SOS and FRS2 binding using respective antibody. The immunoblot was also re-probed for Grb2 as a loading control. The data presented are representative of three independent experiments.

Mentions:
We have shown two independent mechanisms of Grb2 dissociation, both dependent on tyrosine kinase activity. Both these mechanisms could play a fundamental role in Grb2-mediated intracellular signalling. Since Grb2 is known to bind to its downstream effector SOS1, we postulate that only mGrb2 elicits downstream effector function and hence the dimeric Grb2 (dGrb2) is incapable of signal transduction. We tested this hypothesis by investigating the downstream signalling effects of mGrb2 versus dGrb2 in mammalian cells. In cells overexpressing high concentrations the population of Grb2 is expected to be predominantly dGrb2, whilst the dimerization-defective Grb2 mutants will be monomeric regardless of their intracellular concentrations. Cells were transfected with WTGrb2 and Y160EGrb2, and downstream MAPK signalling was compared. The results show that overexpression of WTGrb2 leads to a significant decrease in FGF2- and EGF-induced extracellular signal-regulated kinases (ERK) phosphorylation. An equivalent level of dimerization-defective mutant overexpression on the other hand retains ligand-induced ERK activity (Fig. 4a and Supplementary Fig. 5a). We also confirmed Grb2 concentration-dependent MAPK inhibition by serially increasing in the intracellular Grb2 protein concentration in cells. A direct correlation between increased Grb2 concentration and decreased MAPK was observed (Supplementary Fig. 5c–e). Since the formation of the Grb2–SOS complex is pivotal for RTK-stimulated Ras/MAP kinase activation, we investigated whether the differential complex formation by dGrb2 versus mGrb2 with SOS1 could explain the observed suppression of ERK phosphorylation. Grb2 was affinity purified from cells and the resulting co-precipitating complex was analysed for SOS1 (Fig. 4b and Supplementary Fig. 5b). The results show that the monomeric Y160EGrb2 and N188/214DGrb2 form stable complexes with SOS1 whereas the dimeric WTGrb2 does not. As a control the immunoblot was re-probed with anti-FRS2 antibody, which showed that both dGrb2 and mGrb2 were equally proficient in precipitating the upstream adaptor protein required for FGF receptor signalling.

Bottom Line:
Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome.Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process.Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers.

ABSTRACTThe adaptor protein growth factor receptor-bound protein 2 (Grb2) is ubiquitously expressed in eukaryotic cells and involved in a multitude of intracellular protein interactions. Grb2 plays a pivotal role in tyrosine kinase-mediated signal transduction including linking receptor tyrosine kinases to the Ras/mitogen-activated protein (MAP) kinase pathway, which is implicated in oncogenic outcome. Grb2 exists in a constitutive equilibrium between monomeric and dimeric states. Here we show that only monomeric Grb2 is capable of binding to SOS and upregulating MAP kinase signalling and that the dimeric state is inhibitory to this process. Phosphorylation of tyrosine 160 (Y160) on Grb2, or binding of a tyrosylphosphate-containing ligand to the SH2 domain of Grb2, results in dimer dissociation. Phosphorylation of Y160 on Grb2 is readily detectable in the malignant forms of human prostate, colon and breast cancers. The self-association/dissociation of Grb2 represents a switch that regulates MAP kinase activity and hence controls cancer progression.